Formation of dimeric Fabs in Escherichia coli: effect of hinge size and isotype, presence of interchain disulphide bond, Fab' expression levels, tail piece sequences and growth conditions.
We have made hinge variants of two human Fab's in order to investigate the factors involved in the formation of dimeric Fab's in the periplasm of E. coli. Hinges containing one or more copies of the IgG1 hinge with various numbers of spacing residues were tested. Fab's with hinges based on the gamma2, gamma3 and gamma4 isotypes were also tested. We find that the IgG1 hinge sequence can form approximately 35% F(ab')2 in vivo in shake flask experiments, but that only (approximately) 5% F(ab')2 can be produced during fermentation. IgM and IgA tail-pieces added to Fab's did not effect their multimerisation. The possible role of growth conditions upon F(ab')2 formation in vivo is discussed.
Humphreys
D P
DP
Celltech Therapeutics, Slough, Berkshire, UK. dhumphre@celltech.co.uk
Chapman
A P
AP
Reeks
D G
DG
Lang
V
V
Stephens
P E
PE
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007070:Immunoglobulin A; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D008969:Molecular Sequence Data
D004220:Disulfides; D007070:Immunoglobulin A; D007140:Immunoglobulin Fab Fragments; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D003545:Cysteine